LID scientists are collaborating with scientists from MedImmune under a CRADA to generate candidate vaccines against avian influenza viruses of each subtype, including H5N1 viruses that have caused human infections since 2003. The vaccines were generated using plasmid based reverse genetics and each contains the hemagglutinin and neuraminidase genes from an avian influenza virus and six internal gene segments from the AA ca virus. Based on promising preclinical data in mice and ferrets, clinical lots of vaccines were generated and Phase I clinical trials of the safety and immunogenicity of the vaccines for healthy adults were undertaken under an IND. Several factors contribute to the possibility of a H2 influenza pandemic. First, H2 viruses have proven capable of causing disease in humans. Of the sixteen HA and nine NA subtypes, only H1, H2 and H3 viruses have been known to lead to human influenza pandemics since influenza viruses were first isolated in 1933. While H1N1 and H3N2 viruses have been co-circulating for the past 30 years, H2N2 viruses have not circulated in humans since 1968. Thus, there is declining pre-existing immunity to H2 influenza viruses in the population today. Although H2 influenza has not circulated in humans since 1968, this subtype is maintained in avian reservoirs worldwide. The cold adapted (ca) influenza A/AA/6/60 (AA) (H2N2) virus is the backbone for the live attenuated trivalent seasonal influenza vaccine licensed in the United States. However, only a small number of pre-clinical and clinical trials have been conducted evaluating the AA ca (H2N2) virus as a vaccine. We therefore evaluated the attenuation, immunogenicity and efficacy of the AA ca (H2N2) virus in mice and ferrets in order to determine the suitability of developing this virus as a vaccine candidate for use in the event of an H2 pandemic. The AA ca virus was restricted in replication in the respiratory tract of mice and ferrets. In mice, 2 doses of the AA ca vaccine elicited a greater than four-fold rise in hemagglutination-inhibition (HAI) titer and conferred complete protection against homologous wild type virus challenge. In ferrets, a single dose of the AA ca vaccine elicited a greater than four-fold rise in HAI titer and conferred complete protection against homologous wild type virus challenge in the upper respiratory tract. In both mice and ferrets, the AA ca virus provided significant protection from replication in the upper and lower respiratory tract of heterologous H2 viruses A/Mallard/NY/ 6750/78 (H2N2), A/Japan/305/57 (H2N2) and A/Swine/MO/4296424/2006 (H2N3). The AA ca vaccine is safe, immunogenic and efficacious against homologous and heterologous challenge in mice and ferrets, supporting the evaluation of this vaccine in clinical trials. LID and MedImmune scientists evaluated the immune responses to ca vaccine viruses generated under this program in animal models. Primary and recall B cell responses to live attenuated H5N1 vaccine viruses were examined using a sensitive antigen-specific B cell ELISpot assay to investigate the effect of preexisting heterosubtypic influenza immunity on the development of H5N1-specific B cell immune responses in ferrets. Live attenuated H5N1 A/Hong Kong/213/03 and A/Vietnam/1203/04 vaccine viruses induced measurable H5-specific IgM and IgG secreting B cells after intranasal vaccination. However, H5-specific IgG secreting cells were detected significantly earlier and at a greater frequency after H5N1 inoculation in ferrets previously primed with trivalent live attenuated influenza (H1N1, H3N2 and B) vaccine. Priming studies further revealed that the more rapid B cell responses to H5 resulted from cross-reactive B cell immunity to the hemagglutinin H1 protein. Moreover, vaccination with the H1N1 vaccine virus was able to induce protective responses capable of limiting replication of the H5N1 vaccine virus to a level comparable with prior vaccination with the H5N1 vaccine virus without affecting H5N1 vaccine virus induced antibody response. Our data indicated that previous vaccination with seasonal influenza vaccine may accelerate onset of immunity by an H5N1 ca vaccine which may be of value for pandemic preparedness. Transmission of H5N1 influenza viruses from birds to humans poses a significant public health threat and LID scientists collaborated with scientists at the University of Washington to study the host response of mice to H5N1 viruses. A substitution of glutamic acid for lysine at position 627 of the PB2 protein of H5N1 viruses has been identified as a virulence determinant. We utilized the BALB/c mouse model of H5N1 infection to examine how this substitution affected virus-host interactions and led to systemic infection. Mice infected with H5N1 viruses containing lysine at amino acid (aa) 627 in the PB2 protein exhibited increased severity of lesions in the lung parenchyma and the spleen, increased apoptosis in the lungs, and a decrease in oxygen saturation. Gene expression profiling revealed that T cell receptor activation was impaired at 2 days post-infection (dpi) in the lungs of mice infected with these viruses. The inflammatory response was highly activated in the lungs of mice infected with these viruses and was sustained at 4 dpi. In the spleen, immune-related processes including NK cell cytotoxicity and antigen presentation were highly activated by 2 dpi. These differences were not solely attributable to differences in viral replication in the lungs, but also to inefficient viral clearance early in infection. The timing and magnitude of the immune response to highly pathogenic influenza viruses was critical in determining the outcome of infection. Disruption of these factors by a single aa substitution in a polymerase protein of an influenza virus was associated with severe disease and allowed the virus to spread to extrapulmonary sites.